Regulated polymerization of actin is essential for many cellular processes such as endocytosis, motility and intracellular transport. Actin related protein (Arp) 2/3 complex is the only known nucleator of branched actin filaments. In order to initiate a branch, Arp2/3 complex requires ATP, a nucleation promoting factor (NPF), actin monomers and a mother filament. The VCA (veroprolin homology, connecting motif, acidic) region of NPFs can bind both Arp2/3 complex and actin monomers. During branch formation, Arp2/3 complex must undergo significant conformational changes, allowing the Arp2 and Arp3 subunits to mimic the first two actin subunits in a filament. Actin monomers then elongate from the blunt end of the daughter filament. CK-666 and CK-869 represent two classes of small molecule inhibitors identified to inhibit Arp2/3 complex. CK-666 binds between the Arp2 and Arp3 subunits and appears to prevent the conformational change required for Arp2 and Arp3 to mimic two actin subunits of a filament. CK-869 binds in a hydrophobic cleft of Arp3, propping open the sensor loop in the nucleotide binding pocket. Preliminary experiments, including analytical ultracentrifugation to characterize the size and shape of the complex, support the hypothesis that the inhibitors block the activating conformational change of the Arp2/3 complex. In order to determine the mechanism of inhibition by these compounds, we will utilize previously developed as well as novel methods to assay each step in the pathway of actin filament branch nucleation. Previously developed assays for ATP, VCA, actin monomer, and mother filament binding will be used to determine the effects of the inhibitors on sub-steps of branch formation. Additionally, we will develop new methods such as inter-subunit cross-linking of Arp2 and Arp3 subunits in order to specifically address the effects of the inhibitors on conformational changes of the complex that occur during branch formation. We will also observe the effects of the inhibitors on Arp2/3 complex function in vivo by observing actin patch dynamics, Arp2/3 complex localization and actin treadmilling associated with Arp2/3 complex catalyzed actin polymerization during endocytosis in S. pombe. The inhibitors will allow us to dissect the pathway of actin patch assembly and internalization in a novel way. The inhibitors most likely block steps in the pathway of branch formation which has not previously been studied through direct inhibition. Thus, we expect to gain novel insights into the mechanism of branch nucleation. Additionally, the inhibitors will provide invaluable tools for studying Arp2/3 complex function in more complicated systems due to the fact that they cause rapid, reversible and specific inhibition of Arp2/3 complex without requiring genetic manipulation. In order to properly interpret phenotypes caused by the presence of the inhibitors however, a detailed understanding of their mechanism of inhibition is required.